Plasma membrane repair is a highly conserved mechanism that appears in nearly every eukaryotic organism, from single cell amoebas to most cell types in the human body. It is a prerequisite for development of complex cellular systems and organelle function, for maintenance of cellular integrity following disruption of the lipid bilayer. An ancient primordial cell would not be able to develop the metabolic resources necessary to produce more sophisticated intracellular organelles if any disruption of the external cell membrane resulted in the death of the cell. While a simple lipid bilayer will reseal through thermodynamic principles, establishment of a cytoskeleton network necessitates that the bilayer bordering the cell be held under some degree of tension. When it is held under tension even small disruptions of a lipid bilayer cannot spontaneously reseal (Togo, T., Krasieva, T. B. & Steinhardt, R. A. A decrease in membrane tension precedes successful cell-membrane repair. Mol Biol Cell 11, 4339-46 (2000)), thus intracellular resealing mechanisms must exist to allow for development of complex cellular systems. Multicellular organisms benefit from the capacity to repair cell membranes, particularly in long lived animals where loss of cellular viability could lead to progression of a disease state, such as the heart and brain where there is limited regenerative capacity.
While membrane repair is a conserved mechanism essential for evolutionary development and maintenance of sensitive organs in humans, the pathways facilitating this process are poorly understood. Little, if anything, is known about the mechanism(s) of how a cell repairs disruptions of the plasma membrane. This is not because membrane repair is not common or unimportant; it is simply due to the lack of understanding of the cellular and molecular machinery that regulates this process. While some cells cope with damage to the plasma membrane by death and replacement, many previous studies indicate that repair of acute damage to the plasma membrane is an important aspect of normal cellular physiology (McNeil, P. L. & Ito, S. Gastrointestinal cell plasma membrane wounding and resealing in vivo. Gastroenterology 96, 1238-48 (1989); McNeil, P. L. & Steinhardt, R. A. Plasma membrane disruption: repair, prevention, adaptation. Annu Rev Cell Dev Biol 19, 697-731 (2003)), and disruption of this process can result in a number of different diseases, including muscular dystrophy, heart failure and neurodegeneration (Bansal, D. et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 423, 168-72 (2003); Bazan, N. G., Marcheselli, V. L. & Cole-Edwards, K. Brain response to injury and neurodegeneration: endogenous neuroprotective signaling. Ann NY Acad Sci 1053, 137-47 (2005); Han, R. et al. Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury. J Clin Invest 117, 1805-13 (2007)).
Previous studies established the basic framework of the plasma membrane repair response (McNeil, P. L. & Steinhardt, R. A. Plasma membrane disruption: repair, prevention, adaptation. Annu Rev Cell Dev Biol 19, 697-731 (2003)). It is known that this process requires the translocation of intracellular vesicles (Miyake, K. & McNeil, P. L. Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J Cell Biol 131, 1737-45 (1995)) to the injury site through the action of kinesin and myosin motor proteins. These vesicles then fuse with the plasma membrane in a Ca2+ dependent manner to form a repair “patch”, a process similar to the release of neurotransmitters from neurons (Steinhardt, R. A., Bi, G. & Alderton, J. M. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 263, 390-3 (1994)). Thus, this repair process can be divided into discreet steps involving the; 1) sensing of membrane damage, 2) translocation of vesicles to the injury site, and 3) fusion of vesicles with the plasma membrane. However, the molecular machinery involved in the cellular repair process is not well defined.
Recent studies have identified some molecular components of cell membrane repair, particularly those involved in a pathway thought to be specific to striated muscles. One major finding was our recent discovery that MG53, a muscle-specific TRIM family protein (TRIM72), is an essential component of the acute membrane repair machinery (Cai, C. et al. MG53 nucleates assembly of cell membrane repair machinery. Nat Cell Biol 11, 56-64 (2009); Weisleder, N., Takeshima, H. & Ma, J. Mitsugumin 53 (MG53) facilitates vesicle trafficking in striated muscle to contribute to cell membrane repair. Communicative & Integrative Biology 2, In Press (2009); Cai, C. et al. MG53 regulates membrane budding and exocytosis in muscle cells. J Biol Chem 284, 3314-22 (2009); Cai, C. et al. Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin. J Biol Chem 284, 15894-902 (2009)). MG53 acts as a sensor of oxidation to oligomerize and then recruit intracellular vesicles to the injury site for membrane patch formation. We found that MG53 can interact with dysferlin, another protein involved in membrane repair, to facilitate its membrane repair function, and the membrane trafficking function of MG53 can be modulated through a functional interaction with caveolin-3 (Cav3) (Cai, C. et al. MG53 regulates membrane budding and exocytosis in muscle cells. J Biol Chem 284, 3314-22 (2009); Cai, C. et al. Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin. J Biol Chem 284, 15894-902 (2009)). Our data indicate that maintenance of the MG53-dysferlin-Cav3 molecular complex is essential for repair of the muscle cell membrane and that disruption of these interactions can results in muscular dystrophy and cardiac dysfunction.
Our published findings show that elevated MG53 expression within a cell can increase resistance to cellular disruption, however there are obvious hurdles in controlling. MG53 expression in an organism as a therapeutic approach. However, it was also discovered that placing recombinant MG53 protein outside of the cell can increase the capacity of both muscle and non-muscle plasma membranes to reseal following damage. Direct proof-of-concept studies for the therapeutic use of recombinant MG53 as a membrane repair reagent were based on strong in vitro and in vivo animal model studies, which were detailed in two previous patent applications (PCT/US2007/015815 and PCT/US2008/085573). Recombinant MG53 was found to be highly effective at increasing membrane repair in skeletal muscle, cardiac muscle, epithelial cells and several other cell types. These results indicate that isolated MG53 protein can be applied externally to many different cell types and it will target to sites of membrane damage increase membrane resealing, preventing pathology and improving the structure and function of the tissue.
Herein, we present for the first time data indicating that endogenous MG53 can be detected in circulating blood, and that the level of MG53 in blood serum varies in control (i.e., normal) versus disease. As such, MG53 can be used as a diagnostic biomarker for tissue injury. Moreover, targeting serum MG53 can be a potential therapeutic means for treatment of tissue injury in human diseases.